JPS6354283B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a sulfated ribofuranan having anticoagulant activity in the medical field and a method for producing the same. (Prior Art) Thrombosis or hyperlipidemia often occurs in association with diseases such as malignant tumors, arteriosclerosis, diabetes, and nephrotic syndrome. In recent years, with the increase in the above-mentioned diseases, thrombosis and hyperlipidemia have been on the rise. Currently, effective drugs for these treatments include, for example, dextran sulfate and heparin. Dextran sulfate can be used to treat microorganisms such as Leuconostoc mesenteroides.
α-1,6 produced by
It is a sulfate ester of dextran, which is a polymer of D-glucopyranose, which has anticoagulant effects and is known as an effective drug for treating thrombosis. [Formula] R=H: Dextran R=SO ₃ Na: Dextran sulfate On the other hand, heparin, a mucopolysaccharide present in animal tissues, has a wide range of physiological effects, including strong anticoagulant effects and lipid serum clarifying effects. Although its activity is much stronger than that of artificial heparinoid, the quality of the standard preparation is not constant, and its structure is complex, making the isolation method complicated.
Heparin is characterized by having a sulfated amino sugar in its molecule. The active site in heparin is represented by the following formula. Focusing on the anticoagulant effect of heparin, attempts to treat the surface of materials have been made using GBH (graphite).
benzalkonium chloride-heparin) method.
The GBH method is a method in which benzyconium chloride, a surfactant, is adsorbed onto the surface of a material coated with graphite, and heparin is bonded to it. Similar to the GBH method, the TDMAC method is also used to ionically bond heparin using a surfactant (tetramethylammonium chloride).
Compared to the GBH method, the TDMAC method is unique in that it can be applied to flexible general-purpose polymers such as polyethylene and polyvinyl chloride. Heparin ionically bound to the surface of such materials is eluted into the blood and exhibits anticoagulant activity, so it is used clinically as an antithrombotic material for short-term use (within 24 hours). For example, a material in which heparin is bonded to the surface of polyvinyl chloride using the TDMAC method is used as a bypass blood vessel during surgery. However, it is not suitable for long-term use because the elution rate of heparin into the blood is fast. In addition to these methods, there is a method in which heparin is bound inside the polymer matrix, and the heparin eluted from the surface of the material into the blood is replenished by being released from within the material, thereby sustaining antithrombotic properties. The polymer used in this method is
Some drugs have been confirmed to exhibit antithrombotic properties for 4 weeks or more in in vivo tests. (Problems to be Solved by the Invention) However, these methods have two problems. Since heparin is extracted from animal tissue, the quality of the standard product is not constant, the structure is complex, and the isolation method is complicated. Furthermore, the method in which heparin is eluted into the blood is not suitable for long-term use. (Means for Solving the Problems) In order to solve these problems, the present invention attempts to chemically synthesize heparinoid having high anticoagulant activity like heparin. To this end, the present invention synthesized a novel heparinoid sulfated polysaccharide having an α-1.5-linked sugar chain and containing a sulfate group such as heparin in its molecule. The present invention is based on the following formula (where n is an integer from 10 to 500 and R=H or _SO3Na ). n in the formula is 10-500. When n is less than 10, the anticoagulant activity is small, and when n is less than 10,
If it is larger than , it becomes difficult to synthesize this compound. The present invention also provides a method for producing sulfated ribofuranan by sulfating ribofuranan. As the sulfating agent, anhydrous trimethylamine sulfate complex, chlorosulfonic acid, piperidine N-sulfuric acid, etc. can be used. Furthermore, the present invention resides in a blood coagulation inhibitor containing this sulfated ribofuranan as an active ingredient. Since sulfated ribofuranan has anticoagulant activity, substances containing it as an active ingredient are used as medical polymer materials that need to inhibit blood coagulation, such as in artificial hearts and blood vessels. Ribofuranan, which is a raw material for sulfated ribofuranan, can be obtained, for example, by the following process. Hereinafter, the present invention will be explained in detail based on examples. (Example) Preparation of (1→5)-α-D-ribofuranan (5) D-ribose (1) was subjected to vacuum pyrolysis (Chem, Ber.
106, 3565 (1973)) is used. First, 10 g of sodium hydride was suspended in 100 ml of dry dimethylformamide (DMF), and the above compound
(2) Dissolve 10g in 100ml of DMF and add dropwise while stirring. After reacting for 1 hour, 31 ml of benzyl chloride dissolved in 50 ml of DMF was added dropwise.
Stir at room temperature for approximately 20 hours. The reaction mixture was poured into a large amount of ice water, extracted with chloroform, concentrated, and the compound 1,4-
Anhydro-2,3-di-O-benzyl-α-D
- Obtain 20 g (yield 84%) of ribopyranose (3) crystals. The physical properties of compound (3) are as shown below. mp: 65.0-66.5℃ Specific rotation: [α] ²⁵ _D -35.9° (C1, chloroform) ¹³ C-NMR: δ128.21-138.40 (aromatic), 100.47
(1C, C-1), 82.37 (1C, C-3), 80.22
(1C, C-2), 78.57 (1C, C-4), 73.30,
73.01 (2C, C H ₂ C ₆ H ₅ ), 64.91 (1C, C-5) The obtained compound (3) (3.7 g, 11.9 mmol)
Vacuum dried under high vacuum at 10 ^-5 mmHg overnight and dissolved in methylene chloride (15 ml) previously dried over calcium hydride in a vacuum ampoule. After the polymerization tube is cooled with liquid nitrogen and the monomer solution is sufficiently frozen, boron trifluoride etherate (30μ
, 0.24 mmol). Separate the polymerization tube from the vacuum line and place it in an ethanol bath at -40℃ for 1
Shake vigorously for ~2 minutes. After reacting at -40℃ for 30 minutes, open the polymerization ampoule and pour methanol to stop the reaction. At this time, the polymer precipitates, so chloroform is added to it until the polymer is sufficiently dissolved, neutralized with an aqueous sodium bicarbonate solution, washed with water, and dried over anhydrous sodium sulfate.
After separately removing the desiccant, the liquid is concentrated and petroleum benzine is added for reprecipitation. The operations of dissolving, concentrating, and reprecipitation were performed two more times, and the polymer was dissolved in benzene and freeze-dried to obtain 2,3-di-O
-Benzyl-(1→5)-α-D-ribofuranan
(4) Obtain 3.56 g (conversion rate 96%). The physical properties of polymer (4) are as follows. Specific optical rotation: [α] ²⁵ _D +153.4° (C1, chloroform) n = 1.28 × 10 ⁵ (n = 410) 1,2-dimethoxy 6.0 g of the obtained polymer (4) was dried with metallic sodium in advance Dissolve in ethane (120ml). This solution was added dropwise to liquid ammonia (400 ml) and metallic sodium (5.3 g) kept at -78°C under a nitrogen stream. The reaction system was stirred at -78°C, and after 1.5 hours, ammonium chloride was added until the blue color of the reaction system disappeared. Add more water (10 ml) and evaporate the ammonia at room temperature. Add water (20ml) and add methylene chloride to
Wash twice and dialyze the aqueous layer for 3 days. The aqueous solution was concentrated and lyophilized to form a polymer (1→5)-α-D.
- Obtain 1.35 g (yield 53%) of ribofuranan (5). The physical properties of polymer (5) are as follows. Specific optical rotation: [α] ²⁵ _D +164.1° (C1, water) ¹³ C-NMR: δ102.93 (1C, C-1), 83.56,
(1C, C-4), 71.47 (1C, C-2), 70.37
(1C, C-3), 68.59 (1C, C-5). Preparation of sulfated (1→5)-α-D-ribofuranan of the present invention 0.2 g of polymer (5) was dissolved in pre-dried dimethyl sulfoxide (180 ml), piperidine-N-sulfuric acid (3.0 g) was added, 80 while stirring
Incubate at ℃ for 1 hour. Neutralize with 10% excess of 2.5N aqueous sodium hydroxide and concentrate. The precipitated polymer was dissolved in water by adding methanol, followed by dialysis, concentration, and lyophilization to obtain 0.17 g of sulfated (1→5)-α-D-ribofuranan, a polymer. The physical properties of this polymer are as follows. Metachromatic reaction: + n = ^8.19 there was). Dissolve the test substance in physiological saline to a concentration of 160γ/ml. Also, standard heparin (160 IU/mg)
Prepare 10γ/ml saline. Test substance solution and standard heparin solution each at 0.8,
Transfer 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 ml into glass test tubes (13 x 105 mm), add physiological saline to a total volume of 0.8 ml, and mix. Add 1 ml of bovine plasma* to each test tube and mix, then add 0.2 ml of 2% calcium chloride aqueous solution,
Immediately mix by gently inverting the test tube. After 7-10 minutes, the coagulation status of each test tube was adjusted to 0, 0.25,
The test substance was recorded in classes of 0.5, 0.75, and 1.0, and the titer of the test substance was determined from the amount of the test substance and standard heparin when the coagulation status was 0.5. The results are shown in Table 1. *Bovine plasma: Add 40 ml of 10% sodium citrate (Na ₃ C ₆ H ₅ O ₇・2H ₂ O) aqueous solution to a blood collection container in advance.
Pour 960 ml of fresh bovine blood into this container, mix and centrifuge at 3000 rpm for 10 minutes to collect plasma. [Table] It is known that the sulfated (1→5)-α-D-ribofuranan of the present invention has an anticoagulant activity of about 11% of heparin and about 2.7 times that of the control dextran sulfate. It will be done. In addition, the acute toxicity (LD ₅₀ ) using mice is
It was 1g/Kg or more (intravenous injection). (Effect of the invention) Sulfated (1→5)-α-D-ribofuranan of the present invention and sulfated (1→5)-α-D as a comparative example
-The anticoagulant activity of xylofuranane and dextran sulfate is shown in Table 2. [Table] [Table] Comparing sulfated polysaccharides with approximately the same molecular weight, sulfated ribofuranan has approximately three times the activity of dextran sulfate, but is slightly less active than sulfated xylofuranan. However, critical differences occur during the synthesis routes of ribofuranan and xylofuranane, especially in the monomer synthesis. That is,
Anhydrous xylose, the raw material for xylofuranane, is syrupy, so it is acetylated and purified using silica gel column chromatography, deacetylated, benzylated, purified using column chromatography, and subjected to GPC preparative separation to synthesize the monomer. On the other hand, anhydrous ribose, which is the raw material for ribofuranan, has good crystallinity, so it can be purified by recrystallization, and benzylated products can also be crystallized easily. Therefore, monomer synthesis is much easier for ribose than for xylose. Furthermore, the present invention not only enables the chemical synthesis of heparinoid having anticoagulant activity, but also the heparinoid, which is purely chemically synthesized, has a constant structure and can control physiological activity to a certain extent. moreover,
It is also possible to incorporate heparinoids into the polymer backbone by covalently linking them with synthetic polymers such as segmented polyurethanes to synthesize composites with sustained antithrombotic properties. Furthermore, the present invention can be applied to medical materials that require antithrombotic properties, such as artificial hearts and artificial blood vessels.

Claims (1)

[Claims] 1. General formula: (In the formula, n is an integer from 10 to 500, and R=H or SO ₃ Na). 2 General formula: (In the formula, n is an integer from 10 to 500, and R=H or SO ₃ Na). Manufacturing method. 3 General formula: (In the formula, n is an integer of 10 to 500, and R=H or SO ₃ Na.) A blood coagulation inhibitor containing sulfated ribofuranan as an active ingredient.